The Design of a PNA Probe to Improve Legionella pneumophila Monitoring

Legionella is a significant public health threat in engineered water systems, requiring rapid and accurate environmental monitoring to prevent outbreaks. Traditional detection methods, such as culture-based assays and PCR, are limited by long processing times and the potential for false negatives due to viable but non-culturable (VBNC) cells. This study addresses these limitations by developing a novel Peptide Nucleic Acid-Fluorescence in situ Hybridization (PNA-FISH) method for the specific and sensitive detection of Legionella pneumophila. In silico analysis predicted high theoretical specificity (100.0%) and sensitivity (99.8%), results that were confirmed by experimental validation against 17 L. pneumophila strains and 37 non-target strains (including Pseudomonas, Acinetobacter, and other Legionella species), demonstrating strong fluorescence signals with no cross-reactivity. Furthermore, the method was successfully applied to artificially contaminated tap water, achieving a limit of detection of 103 CFU mL−1 directly on the filter membrane. This work highlights the potential of PNA-based probes to improve bacterial monitoring, offering fast, reliable, and field-adaptable detection of L. pneumophila. The findings support the integration of this probe into routine water system monitoring workflows, facilitating timely assessment of contamination and outbreak prevention.